Revisiting the bromination of 3β-hydroxycholest-5-ene with CBr4/PPh3 and the subsequent azidolysis of the resulting bromide, disparity in stereochemical behavior

Cholesterol reacts under Appel conditions (CBr4/PPh3) to give 3,5-cholestadiene (elimination) and 3β-bromocholest-5-ene (substitution with retention of configuration). Thus, the bromination of cholesterol deviates from the stereochemistry of the standard Appel mechanism due to participation of the Δ5 π-electrons. In contrast, the subsequent azidolysis (NaN3/DMF) of 3β-bromocholest-5-ene proceeds predominantly by Walden inversion (SN2) affording 3α-azidocholest-5-ene. The structures of all relevant products were revealed by X-ray single crystal structure analyses, and the NMR data are in agreement to the reported ones. In light of these findings, we herein correct the previous stereochemical assignments reported by one of us in the Beilstein J. Org. Chem. 2015, 11, 1922–1932 and the Monatsh. Chem. 2018, 149, 505–517.

Introduction 3β-Hydroxycholest-5-ene (cholesterol) is a structural and physiologic amphipathic steroid in human and animals as well. Cholesterol is an essential component of the plasma membrane, where it acts as fluidity buffer, permeability switch, and consequently in cell signaling pathways. Physiologically, cholesterol is the substrate for the biosynthesis of steroidal hormones, vitamin D and bile acids [1,2].
Diets of animal sources like red meat, liver, milk, and butter provide the body with its daily needs of cholesterol. In addition, hepatocyctes synthesize cholesterol through the mevalonate pathway. Dietary cholesterol is absorbed into the blood stream through a specific membrane bound protein named Niemann-Pick C1-Like 1 (NPC1L1) on the gastrointestinal tract epithelial cells as well as in hepatocytes. As hydrophobic molecule, it circulates in the blood stream engulfed in carrier lipoproteins of two types, high density lipoproteins (HDL) or good proteins and low-density lipoproteins (LDL) or bad proteins [4,5].
People with a total blood cholesterol over 125-200 mg/dL are considered hypercholesterimic. They are under high risk of cholelithiasis (formation of gallstones), atherosclerosis, heart attack, stroke, peripheral artery disease, and cancer [4,5]. Synergistic cholesterol lowering medications are inhibitors of cholesterol absorption (ezetimibe) and cholesterol biosynthesis (statins). However, the side effects of these drugs are controversial. Therefore, synthetic cholesterol derivatives came into focus for recent applications in chemical biology and materials science [6]. The advances have been summarized in comprehensive reviews [7,8].
In previous studies, one of us (M. R. E. A.) felt intrigued by the potential of chemical hybridization of cholesterol through simple connections of pharmacophores including sugars, chalcones, quinolone, theophylline, and ferrocene using click chemistry [9][10][11]. Following this strategy, cholesterol was propargy-lated, coupled with azido quinoline, and then functionalized with glucose as part of random designs to discover new antimicrobial and cytotoxic candidates. From these studies, conjugates I [9] and II [10] were identified to display an excellent preliminary antibacterial impact, and congener III [10] showed a good cytotoxic effect against the prostate cancer cell line PC-3 ( Figure 2). When the spacer of I was increased from C 6 to C 11 , the antimicrobial potential dramatically decreased [11].
In order to extend the compound platform, the synthesis of 3-azidocholest-5-ene was addressed [10]. Starting from natural cholesterol, a double inversion of the stereogenic centre at C3 through an Appel type two step conversion of cholesterol into the 3-azido derivative via the corresponding bromide was assumed. Thus, the expected product was 3β-azidocholest-5-ene [10]. Lacking crystallographic evidence, the synthetic chemistry was expanded to click conjugates such as II and III, and the data was reported [10].
Recently, those studies were revisited, and we now obtained single crystals which allowed to unequivocally establishing the relative configurations of the products by X-ray crystallography. Accordingly, the stereochemistry at C3 of the bromo-, azido-and triazolocholesterols was incorrectly assigned, and we now wish to correct the previous reported structures.

Results and Discussion
3β-Hydroxy-Δ 5 -steroids, for instance, cholesterol, pregnenolone, and their derivatives which possess a potential leaving group at the 3β-position, have a unique feature in their chemical reactions. In these steroids, the breaking of the C3-X bond is facilitated by the formation of a cationic strained cyclopropane intermediate, which is formed by translocation of the C5-π bond electrons to the homoallylic carbon atom at C3 [12]. In this way, substitutions at the stereogenic homoallylic carbon atom can proceed with retention of configuration. Concurrently, a so-called i-steroid rearrangement leads, for instance, to 6β-azido-3α,5-cyclo-5α-cholestane by 6β-face attack of the steroidal substrate by the nucleophile [13,14].
In similar work, Peterson and co-workers reported several examples of such stereoretentive conversions of cholesterol, which provided the corresponding 3β-halo-and 3β-azido-5cholesterenes in high yields [12]. The cholesterol mesylate was the most effective intermediate, and the nucleophiles were trimethylsilyl-based nucleophiles. TiCl 4 and BF 3 ·OEt 2 served as activators. No reaction was observed with the 3α-mesyl analog and the cholestane congener. The 3β-azido derivative could also be obtained from 6β-azido-3α,5-cyclo-5α-cholestane [14] by treatment with a mixture of TMSN 3 and BF 3 ·OEt 2 [12]. All of those results confirmed the involvement of regio-and stereospecific i-steroid and retro-i-steroid rearrangements. Later, tetrabutylammonium halides were used as cost effective and stable alternatives of TMS-based reagents [15]. Treatment of compound 4 (Scheme 1) with NaN 3 in refluxing toluene was re-ported to proceed with retention of configuration to afford the β-epimer 6 [16]. Another nice application of this chemistry was recently reported by Oestreich and co-workers, who converted 3β-hydroxypregn-5-en-20-one into the corresponding 3-bromo derivative, which also occurred with retention of configuration at C3 [17].
In 2008, a direct dehydroxyazidation of cholesterol by treatment of the steroid with a zinc azide-pyridine complex, diisopropyl azodicarboxylate (DIAD), and PPh 3 was described [18]. This Mitsunobu-like reaction occurred with complete inversion at C3 to afford 3α-azidocholest-5-ene (5) in high yield. The same product was recently obtained by direct dehydroxyazidation of cholesterol upon treatment with N-acetyl azidobenziodazolone (ABZ) and PPh 3 in THF [19], Table 1.
While synthesizing new potential biologically active probes with cholesterol scaffolds in the Port Said laboratories, particularly from 3-azidocholest-5-ene, we started wondering about the previously reported structural and stereochemical assignments of the steroid derivatives. After repeating the C-OH to bromide exchange of cholesterol (1) under Appel conditions, we now   [14] found two products in different yields, 4 (80%), and 9 (8%), Scheme 2. Their polarities were so similar that they merged during color development on the hot TLC plate.   The less polar, minor material gave ice-white needles, and an X-ray single crystal structure determination revealed the product to be cholesta-3,5-diene (9, Figure 3).
The major, slightly more polar product of the Appel reaction was 3β-bromocholest-5-ene (4, Figure 4). It crystallized as colorless plates, and the structural and stereochemical assignment of 4 was unequivocally confirmed by X-ray crystal struc-ture determination. Compound 4 was erroneously reported to have the α-configuration at C3 [10]. Now, the NMR chemical shift data of 4 are in full agreement with those reported earlier [12].
Comparing the respective NMR data with the published ones [12] revealed a difference of about 0.6 ppm for the chemical shift of H3 suggesting a miss-assignment [23]. This assumption was confirmed by the X-ray structure analysis of single crystals obtained from Et 2 O, which showed the product to be 3α-azido- cholest-5-ene (5, Figure 5) [18,19]. Thus, under the aforementioned conditions, 3β-bromocholest-5-ene (4) was predominantly converted into 3α-azidocholest-5-ene (5) involving a stereospecific transformation at C3 proceeding with a Walden inversion. For note, the 1 H NMR spectrum of 5 revealed the presence of ca. 15% of the β-epimer 6, which could result from an incomplete stereospecificity of the substitution opening an alternative reaction path. Also in this case, the NMR data are then in agreement with the reported ones [18].
In light of these results, the mechanistic interpretation depicted in Scheme 2 can be provided. Under Appel conditions with a combination of CBr 4 and PPh 3 , 3β-hydroxycholest-5-ene (1) leads to two products, cholesta-3,5-diene (9) and 3β-bromocholest-5-ene (4). Both 9 and 4 result from intermediate 10, in which the C3 hydroxy of 1 is activated. Deprotonation of 10 at C2 with bromide as base provides diene 9 as the minor product. Bromide 4 is formed via cyclopropyl cation 11, which is generated from 10 by loss of triphenylphosphine oxide being supported by involvement of the Δ 5 π-bond electrons from the α-face. Stereospecific reaction of 11 with bromide as nucleophile leads to 4, in which the halo substituent is located on the β-side of the molecule. Treatment of 4 with NaN 3 in DMF at 90-100 °C provides predominantly azide 5 [23]. This reaction has a high stereospecificity as well, proceeding mostly with inversion of configuration at C3 (Walden inversion). Consequently, the newly introduced substituent is located on the α-face of the steroid. Interestingly, this result contrasts the one observed when 3β-mesylcholest-5-ene is treated with TMSN 3 / BF 3 ·OEt 2 [12]. There, the process proceeds by retention of con-figuration locating the azido substituent on the β-face of the steroid (compound 6).

Conclusion
For each product 4, 5 and 9 the stereochemical assignment has now been confirmed by X-ray single crystal structure determination, and the NMR data are in agreement with those of previous reports. Former structural interpretations of 4, 5 and 9 as well as those of follow-up compounds [10,11] need to be corrected as shown in (Figure 6).

Figure 6:
Compounds (next to 4, 5 and 9) to be corrected in refs. [10] and [11]. The respective bonds are highlighted in red.

Supporting Information
Supporting Information File 1 X-ray crystallography and NMR spectra.

Supporting Information File 2
Crystallographic information files for compounds 4 and 9.